When it binds Ca2+, calmodulin (CaM) activates an enormous array of different enzymes with critical functions in the cell. Our preliminary results indicate the maximum global free concentration of Ca2+-CaM in cells is approximately 30 nM, and the total concentration of CaM is well below the concentration of CaM-binding proteins. While many CaM-dependent enzymes bind Ca2+-CaM with dissociation constants of approximately 1 nM, some have affinities hundreds of times lower. Among this group are the CaM-dependent adenylate cyclases, which are robustly activated by Ca2+ in cells. This is not consistent with the low global free concentrations of Ca2+-CaM that we have measured. However, conditions that produce local increases in the free Ca2+ concentration at the plasma membrane appear to be required to significantly activate the cyclases in several cell types. And they co-localize in the brain with neuromodulin or neurogranin, membrane-associated proteins thought to function as CaM "sinks". We have therefore hypothesized the existence of two mechanisms that enhance the free Ca2+-CaM concentrations at the plasma membrane to produce the observed activation of the cyclases: (1) Diffusional recruitment of CaM due to local elevations in Ca2+, and (2) Concentration of CaM at the plasma membrane by CaM sink proteins. We propose to test this hypothesis by determining how the free concentrations of Ca2+-CaM produced at the plasma membrane are affected by local elevations in the free Ca2+ concentration and expression of neuromodulin, and how this relates to changes in the activity an expressed CaM-dependent cyclase activity (AC1). We also,,will investigate whether PKC- catalyzed phosphorylation of neuromodulin, which abolishes its CaM sink function, can dynamically control free Ca2+-CaM concentrations and cyclase activity. Given the limiting amount of CaM in the cell, it is clearly of general importance to understand how it is distributed among CaM-dependent enzymes, and how this impacts cellular function. This proposal emphasizes factors that enhance the free Ca2+-CaM concentrations at the plasma membrane and control the activities of CaM-dependent cyclases, as well as other low affinity CaM-dependent enzymes. The cyclases are a particularly important class of low affinity CaM targets, having been specifically been implicated in secretion and steroidogenesis in parotid acinar and adrenal glomerulosa cells, and in some forms of learning.